The aim of this study was to assess the effect of three calcium silicate-based sealers  and two epoxy resin-based sealers  on various aspects, such as cell viability, inflammatory response, and osteogenic potential, of human periodontal ligament stem cells (hPDLSCs). AH-Plus showed the lowest cell viability on hPDLSCs in all time periods in fresh media. In set media, hPDLSCs showed no significant differences in cell viability among all the tested materials. Wellroot ST showed the highest level of cell adhesion and the morphology of attached cells. AH-plus presented a significantly higher expression of IL-6 and IL-8 than the other sealers. AD Seal and three calcium silicate sealers showed high expression of the mesenchymal stem cell markers. ALP mRNA expression showed a significant increase in time-dependent manner on all of three calcium silicate-based sealers, which do not seem to interfere with the differentiation of hPDLSCs into osteoblasts. Based on the results from this study, calcium silicate-based sealers appear to be more biocompatible and less cytotoxic than epoxy resin-based sealers. Meanwhile, further and long-term clinical follow-up studies are required. Three-dimensional obturation of the root canal system is an important step in root canal treatment . In the Ideally, the contact between the root canal wall and the filling material is terminated at the physiologic apical foramen area . HoweverHuman periodontal ligament stem cells (hPDLSCs) are a kind of multipotent stem cell population and recently have been frequently used for in vitro biocompatibility study . MeanwhiThe aim of this study was to assess the effect of three calcium silicate-based sealers, EndoSeal MTA, Nano-ceramic Sealer, and Wellroot ST on various aspects, such as cell viability, inflammatory response, and osteogenic potential, of hPDLSCs. The null hypothesis was that the calcium silicate-based sealers and epoxy resin-based sealers had the similar biocompatibility. n = 4) of four healthy subjects, whose ages ranged from 16 to 24 years old. Teeth were extracted under the purpose of orthodontic treatment, and the written informed consent forms according to the guidelines of the Ethics Committee of the research institution  were obtained from the donors. Human PDL tissue was isolated from the middle-third of the root and minced to 2 mm size pieces using a surgical blade. Then, the pieces of hPDL tissue were washed five times using Dulbecco’s modified Eagle’s medium  containing 3% penicillin-streptomycin (Gibco) and placed on a 6-well culture plate . The explants were grown in DMEM with 15% fetal bovine serum  and 1% penicillin-streptomycin. Migrated hPDLSCs from the explants were carried out to passage 2 cells by cell dissociation using trypsin (Gibco). The isolated hPDLSCs were cultured and incubated at 37 °C in 5% CO2. For all experiments, hPDLSCs at passages 4 to 6 were used.hPDLSCs were obtained from premolars  and three calcium silicate-based sealers  were used for the experimental materials .2 at 37 °C. The supernatant of material extraction medium was filtered using a 0.2 μm pore-size syringe filter . A dilution (1/4 vol/vol) of these material extraction media was prepared with culture medium.All experimental sealers prepared according to the manufacturers’ instructions were mixed with culture medium (DMEM) at a concentration of 20 mg/mL to make material extraction medium, incubated for 24 h in 5% CO2 at 37 °C. Every 24 h the medium was changed, and then the 48-h and 72-h extraction media were collected and passed through a 0.2 µm filter  . Set specimens of the tested sealers were made with discs  fabricated from cylindrical Teflon molds in sterile conditions. To prepare an extraction vehicle, each set sample disc was stored and incubated in DMEM solution with 10% FBS and 1% penicillin-streptomycin for 72 h in 5% COGermany) . 4) were seeded in a 24-well plate and exposed to the extraction media  for 7 days. After each experiment period, MTT solution  was added to each well and incubated for 2 h. Subsequently, the medium containing MTT solution was removed, and 300 μL dimethyl sulfoxide  was added to each well to dissolve the MTT formazan. The dissolved formazan solution of each well was transferred to a 96-well plate and measured at 540 nm in microplate reader .The 3--2,5-diphenyltetrazolium bromide (MTT) assay was performed to investigate the influence on the cell viability, proliferation, and the toxicity of all materials on hPDLSCs. The cells (2 × 104 cells/mL) were directly seeded onto each set disc and incubated in 5% CO2 at 37 °C for 72 h. The discs were taken out from 96-well plate, rinsed with PBS and fixed with 2% glutaraldehyde. To examine the cell morphology on the discs using scanning electronic microscopy , all discs were subjected to dehydration, air-drying, and 100-nm-thick gold/palladium coating. The magnifications used were 200× and 1000×. All sealers were made with discs (5-mm diameter and 2-mm thick) fabricated from cylindrical Teflon molds in sterile conditions. After transferring the disc into the 96-well plate (Merck), hPDLSCs (5 × 105 cells) were seeded in a 6-well plate, and then after 24 h the medium was changed to 1/4 diluted fresh extraction medium and incubated for 24 h. The concentrations of IL-1β, IL-6, IL-8, IL-10, and TNF-α in the fresh extraction medium were analyzed using ELISA kit (R&D systems) according to the manufacturer’s recommendations and measured at 450 nm in a microplate reader (BioTek).To evaluate the inflammatory response of all materials, enzyme-linked immunosorbent assay (ELISA) was conducted. hPDLSCs (1 × 105 cells) were seeded in a 6-well plate, and 24 h later, medium was changed to 1/4 diluted fresh extraction medium and incubated in 5% CO2 at 37 °C for 72 h. Then, the cells were harvested using 0.05% Trypsin-EDTA, washed with PBS, and made a single-cell suspension for CD markers antigen-antibody reaction and FACS analysis. The reaction of the CD makers in hPDLSCs was evaluated using BD Stemflow human MSC analysis kit  and performed according to the manufacturer’s instructions. All data were analyzed using FlowJo software (BD Biosciences).To confirm the mesenchymal phenotypic characterization of hPDLSCs, flow cytometry  assay was carried out. The expression of mesenchymal stem cell surface molecules  was analyzed. hPDLSCs (1 × 105 cells) were placed in a 6-well plate, and incubated for 3, 6, and 9 days in the prepared medium as follows: 1/4 diluted fresh extraction medium including with 100 µM L-ascorbic acid 2-phosphate (Merck), 10 mM b-glycerol phosphate (Sigma), and 100 nM dexamethasone (Sigma). The mRNA expression levels of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2) were determined by real time qPCR taking the β-actin encoding gene as internal reference. Isolation of mRNA and transcription into cDNA was performed using the Rneasy mini kit , and revertAid first strand cDNA synthesis kit (Invitrogen). qPCR was performed on QuantStudio 3 Real-Time qPCR systems (Applied Biosystems) in paired reactions using the Taqman gene expression assays ALP, Hs00758162_m1; RUNX2, Hs01047943_m1; β-actin, Hs01060665_g1 . Changes in the expression of target genes were calculated using the 2−ΔΔCt method. To find the effects of three calcium silicate-based sealers  on osteoblastic differentiation, mRNA expression of differentiation markers was evaluated. hPDLSCs were cultured for 3, 6, and 9 days in order to confirm the pattern of osteogenic genes for all sealers, with the results being analyzed using real time qPCR (RT-qPCR). hPDLSCs (1 × 104 cells) were placed in a 24-well plate and incubated for 3, 6, and 9 days in the fresh extraction medium containing osteogenic-inducing reagents . After each time period, cells were fixed with 4% PFA for 10 min, washed twice in PBS, and stained using the ALP Staining Kit (Merck) according to the manufacturer’s recommendations.Alkaline phosphatase (ALP) staining was conducted to measure the osteogenic activity. hPDLSCs (2.5 × 10p < 0.05).Data were analyzed with SPSS software  for the comparison between materials and the test periods. After checking the data normality, statistical significance was evaluated by Friedman test and Dunn post-hoc for the comparison of materials. The differences between test periods were evaluated by Kruskal–Wallis H and post-hoc Bonferroni–Dunn test (p < 0.05)  .p < 0.05)  were used. In the 48-h extraction media, hPDLSCs showed no significant differences in cell viability among all of the tested materials and control for the experimental periods (1 to 7 days) A. In the < 0.05) B. The inSurface analysis on the morphology and adhesion of hPDLSCs on the fully set material disc was performed by scanning electron microscope (SEM). After incubation, hPDLSCs being attached on three calcium silicate set discs  were observed, whereas set surface of AH-Plus and AD Seal discs did not show any cell attached due to the cell death . Wellroop < 0.05) . AH-Plus presented a significantly higher expression of IL-6 and IL-8 than the other sealers ( < 0.05) .hPDLSCs were cultured for 3 days in different types of endodontic sealer eluates . Fresh extraction medium of the AH-plus showed high toxicity in hPDLSCs , therefop < 0.05)  and time periods  A. Howeve < 0.05) B. In add < 0.05) . Contemporary root canal treatment has been advanced not only for the clinical convenience but also for good prognosis from the development of various materials and techniques. Among the newly introduced materials, calcium silicate-based sealers are the most recently introduced representative materials, but there is still a lack of evidence of their characteristics and clinical use. From this study, calcium silicate-based sealers, such as EndoSeal MTA, Nano-ceramic Sealer, and Wellroot ST, appeared to be more biocompatible and less cytotoxic than epoxy resin-based sealers.The calcium silicate-based sealer is usually supplied in a single paste syringe, which clinicians use directly to the root canal by injection . Thus, tCell viability is defined as a percentage of live cells in a whole population and usually used to evaluate the toxicity of materials . VariousInitial toxicity of epoxy resin-based sealers immediately after mixing and before complete set is well known because of the release of the sealer’s amine or epoxy resin components ,17. In tIn the present study, even calcium silicate-based sealers  showed decreased cell viability by time in fresh media, which might be a result of their high pH in the fresh state. Calcium silicate-based sealers have a high alkalinity because they dissolve into calcium hydroxide when in contact with soft tissues . AlthougConsidering all results from this study, the initial contact of periodontal tissue with the fresh mixed sealers would be worse than that with set sealers. In general, clinical situations, the contact area with the sealer is limited around the apical region , which mThe cell viabilities according to the extraction time (48-h and 72-h) from set sealers were also investigated, and it was found that there were no significant differences among all of the tested sealers with increasing extraction time. The cell viability in the 48-h extraction media was not statistically significant by all experimental periods. Even AH-Plus, which was the most toxic in fresh media state, showed decreased toxicity to the same level of the control. This indicates that cytotoxicity of AH-Plus would be decreased after being fully set. In 72-h extracted media after set, Wellroot ST showed significantly higher cell viability than control at 3 days, and the viable cells were significantly increased for 7 days in Nano-ceramic sealer. Therefore, it may be implicated that calcium silicate-based sealers have a good effect on the survival and proliferation of hPDLSCs. However, further studies are needed to clarify intracellular signaling pathways involved in this cell proliferation by calcium silicate sealers.SEM observations in this study showed the death of all hPDLSCs on the surface of AH-Plus and AD Seal set specimen. These findings indicated that two epoxy resin-based sealers have toxicity on hPDLSCs even after having been fully set. On the other hand, the cells were well attached and proliferated on the set surface of three calcium silicate-based sealers, which means that they would be more advantageous to cell biocompatibility even in direct contact conditions than epoxy resin-based sealers. Wellroot ST was found to be the most effective for attachment of hPDLSCs on the set surface. The favorable cell attachment and proliferation on Wellroot ST and Nano-ceramic Sealer might be due to their smooth surface. However, they might have been difficult to be attached and differentiated on EndoSeal MTA, which has too rough a surface. In addition, the ability of cells to attach to the surface of the sealer appears to be influenced by the chemical composition of the sealer .Pro-inflammatory cytokines are involved in the up-regulation of inflammatory reactions. There is abundant evidence that certain pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, are involved in the process of pathological pain . It is kSealers with the ability to enhance osteogenesis have the potential to promote faster and more predictable healing of apical periodontitis. Thus, the evaluation of sealer for these potentials can improve a clinician’s ability to choose an endodontic sealer that will not only minimally inhibit wound healing but also possibly help expedite the process.Mesenchymal stem cells (MSC) are known to be isolated from bone marrow, peripheral blood, placenta, and dental pulp, etc. . The hPDIt is desirable for root canal sealers to present the capacity of modulating the periapical environment . RUNX2 iALP is a factor for evaluating an initial cell differentiation into osteoblasts . RT-qPCRConsidering above the results, the three calcium silicate sealers do not seem to interfere with the differentiation of hPDLSCs into osteoblasts and the expression of osteoblastic markers present in the apical tissues, enabling favorable healing of periapical lesion. In particular, Nano-ceramic Sealer has favorable initial osteoblastic potential, which is more beneficial for initial periapical healing than other sealers.Thanks to the advantageous features of the calcium silicate-based sealers, a new canal-filling technique using single cone with or without compaction has been suggested as an up-to-date alternative to the current standard of warm condensation techniques. Future in vitro or clinical studies would be necessary to strengthen the rationale for the new materials and techniques. Based on the results from this study, calcium silicate-based sealers  appear to be more biocompatible and less cytotoxic than epoxy resin-based sealers.


The present study was conducted to examine the effect of sleep deprivation (SD) on the anti-apoptotic pathways in Parkinsonian rats. Male Wistar rats (n = 40) were assigned to four groups : sham surgery (Sham), 6-hydroxydopamine (6-OHDA)-lesioned (OH), 6-OHDA-lesioned plus grid control (OH+GC), 6-OHDA-lesioned plus SD (OH+SD). Parkinson’s disease (PD) model was induced by the unilateral intra-striatal infusion of 6-OHDA (10 µg/rat). SD  was induced using a multiple platforms water tank. On the last day of interventions, animals were subjected to open field test for horizontal motor performance assessment. Also, brain-derived neurotrophic factor (BDNF), Bcl-2 and Bax were assessed in the striatum of study groups. SD obscured the motor deficits of PD animals observed in open field test. BDNF level and Bcl2/Bax ratio significantly increased in the OH group, and SD reduced their levels in the PD animals. SD suppressed the anti-apoptotic compensatory responses in the striatum; therefore, it may accelerate continual neuronal cell death in PD. Parkinson’s disease (PD) is a progressive and disabling neurodegenerative disorder affecting nearly 1-2% of elderlies. It results in dysfunction of dopaminergic (DAergic) neurons in the nigrostriatal system. This accounts for major PD symptoms including resting tremor, muscle rigidity, and bradykinesia . DegenerAnimals and experimental design °C with free access to food and tap water. Animals were housed at a 12 hr light/dark schedule (light at 7:00 AM). All experimentations were performed during the light cycle. In order to prevent occurrence of social instability, before initiation of the experiment, rats were kept in the same cages for 15 days to stabilize a social hierarchy within the group. This study was confirmed by the Ethical Committee of the Tabriz University of Medical Sciences (TBZMED.REC.1394.838), and all experimental procedures were performed in accordance with the National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals . Male Wistar rats  (180-220 g) were used in this study. Rats were kept in standard polypropylene cages and maintained at constant temperature of 23 Animals were randomly allocated to one of the following groups (n=10 each): sham surgery (Sham), 6-OHDA-lesioned (OH), 6-OHDA-lesioned plus chronic SD (OH+SD), and 6-OHDA-lesioned plus grid control (OH+GC). Rats in the sham group underwent stereotaxic surgery and received 6-OHDA vehicle. OH, OH+SD and OH+GC groups were subjected to the intra-striatal injection of 6-OHDA. Rats in the sham and OH groups were kept in their home cages throughout the study period. Using a modified multiple platforms (MMP) tank, rats in the OH+SD group were subjected to the SD protocol; 4 hr/day for 14 days . The OH+6-OHDA lesionUnilateral lesions of the right striatum was induced by the stereotaxic infusion of 6-OHDA hydrobromide  under ketamine and xylazine anesthesia . The coordinates for this site were determined according to the rat brain stereotaxic coordinates: anteroposterior from bregma (AP) = 0.4 mm, mediolateral from the midline (ML)= 2.8 mm and dorsoventral from the skull (DV)= -5 mm  Figure . DesipraSD protocol°C) until the water surface reached 1 cm below the edge of the platforms. During the SD period, rats had access to unlimited food and water, which had been provided through hung bottles and pellets boxes. Upon entering into rapid eye movement (REM) sleep, rats lose their postural tone and contact with or drop into the water  were put in the MMP water tank . The tank contained 15 platforms (8 cm high and cm 6.5 diameters), horizontally and vertically spaced apart by 13 cm and 10 cm (edge to edge), respectively and arranged in 3 rows such that did not limit rats movement from one platform to another. The tank was filled with water For evaluation of horizontal motor performance, each rat was individually placed in the center of square open field arena (90× 90×45 cm) made of opaque black polyethylene. The locomotor activity of the animals during 5 min was videotaped by a ceiling-mounted video camera, and the total distance movement was analyzed using Noldus tracking software . After each test session, the arena was cleaned carefully with cotton wet with a 10% ethanol solution to remove the presence of any olfactory clues . 6-OHDA-induced lesion verificationRats with 6-OHDA lesion received an acute intraperitoneal injection of amphetamine (2.5 mg/kg) to confirm the efficacy of unilateral lesion of the striatum. Only rats exhibiting at least 5 full (360°) ipsilateral rotations per min were considered as Parkinsonian rats and included for sampling and data analysis .Sampling°C for further processes.At the end of the behavioral experiment, the animals in different groups were euthanized and the brains were quickly removed from the cranium. In all groups, the right hemisphere was separated along the midline and striatum was dissected on ice. This hemisphere was selected for tissue collecting, because in the sham and OH groups, it underwent stereotaxic surgery. The collected striatum was rapidly frozen in the liquid nitrogen and then stored at -80 Isolation of RNA and real time PCR°C until the time of use. Complementary DNA (cDNA) was synthesized using 3 µg of total RNA by Accupower cyclescript RT premix (dn6) . qRT-PCR was performed by a LightCycler 96 - Roche system using 10 µl of SYBR Premix , 0.4 µM of each primer . Total RNA of the right striatum was extracted using AccuZol solution , according to the manufacturers’ instructions. The quality and concentration of the isolated RNA were evaluated using agarose gel electrophoresis and NanoDrop , and samples were stored at -80 h primer , 1 µl oft method , and β-aEnzyme-linked immunosorbent assay for BDNF, Bcl-2 and Bax2PO4 ,1 mmol KCl , 50 mmol Tris-HCl pH 7.4, 1 mmol ethylenediamine tetraacetic acid (EDTA), 1 mmol sodium fluoride (NaF), 1 mmol sodium orthovanadate (Na3VO4) and Triton X100 and protease inhibitors cocktail , using polytron homogenizer  on ice. The homogenized striatum was centrifuged at 4 °C for 15 min at 10000×g to remove the insoluble materials form the specimen. Total protein concentration in the supernatant was determined using Bradford method. The striatal contents of BDNF, Bcl-2 and Bax were measured using commercially available ELISA kits  according to manufacturer’s recommendations. For protein extraction, the striatum tissue was homogenized in the in lysis buffer containing 1 mmol KHStatistical analysispost-hoc test. P<0.05 was considered statistically significant.Statistical analysis was performed using Graph Pad Prism 6.01 . All data were expressed as mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s Striatal BDNF expressionP<0.01). BDNF decreased in the OH+SD group compared to the OH and OH+GC groups (P<0.001). No difference was found in the amount of this protein between the OH+GC and OH groups  and protein (P<0. 001]) ratios in comparison with the OH and OH+GC groups. No difference was found in the Bcl-2/Bax mRNA and protein in the striatum of OH+GC group compared to the OH group (As shown in OH group .OFTP<0.001). Also, induction of SD significantly increased total distance movement in the OH+SD group compared to the OH and OH+GC groups (P<0.05). The traveled distance did not significantly change between OH+GC and OH groups (The traveled distance was significantly lower in the Parkinsonian rats than that of the sham group 14 days post 6-OHDA-lesion induction (H groups .This study showed that exposure to long-term (14 days) SD could attenuate the anti-apoptotic resistance in the 6-OHDA-lesioned rats. We also found that SD relatively obscured motor impairment observed in OFT. Moreover, our results showed that the intra-striatal injection of 6-OHDA (10 μg/rat) resulted in the activation of anti-apoptotic compensatory responses to prevent apoptotic cell death in nigrostriatal pathway. However, these reactions were not completely able to mask motor abnormalities in the Parkinsonian rats. Interestingly, the disruptive effect of SD on the anti-apoptotic compensatory reaction of 6-OHDA-lesioned rats seems to be due to SD alone rather than the stress of the SD technique, because rats in the OH+GC group did not show any changes at the molecular and behavioral levels compared to the OH group. The unilateral 6-OHDA rat model of PD provides quantifiable motor disturbances, and when neurotoxin is injected into the striatum, it results in the retrograde degeneration of the nigrostriatal neurons , 37. TheOur results showed that 6-OHDA lesion increased BDNF content in the striatum. This is in accordance with the previous studies that have demonstrated an increased level of BDNF in PD patients and in the midbrain of 6-OHDA-lesioned rats , 38, 39.Neurotrophic factors including BDNF are extensively distributed in basal ganglia and provide trophic supports to the motor neurons . BDNF biThere is a close relationship between BDNF and sleep regulation, because exogenous BDNF increases sleep duration in different animal species, and SD reduces brain BDNF levels . et al., SD decreases BDNF expression in the hippocampus of the rats  in the striatum of sham rats. Apoptosis is mediated through two distinct members of Bcl-2 family including Bax and Bcl-2. The Bax protein facilitates the release of apoptogenic molecules from the mitochondria. Opposed to Bax, Bcl-2 proteins block mitochondrial leakage of cytochrome c through stabilizing mitochondrial outer membrane and prevent apoptotic cell death , 53, 54.iability . Moreoveiability . Becauseiability .We found that Bax mRNA and its protein levels remain unchanged after 14 days in the 6-OHDA-lesioned rats. This is in line with the reports that showed no alteration in the expression levels of Bax protein in the post-mortem brain tissue of the PD patients . BecauseIn this study, a reduction in the elevated level of striatal Bcl-2 mRNA and protein induced by SD was associated with an increased level of Bax both at mRNA and protein levels in the Parkinsonian rats. Because the ratio of these two members predicts the cells’ tendency to apoptosis, it is probable that SD may cause an imbalance of the Bcl-2/Bax ratio that tilts the scales toward DAergic neurons degeneration in the OH+SD rats.Immunohistochemical investigations on the rat brain revealed that SD reduces Bcl-2 positive neurons and increases the number of Bax expressing neurons, resulting in the neuronal loss via apoptotic pathways . SD has Given these, one possible mechanism for reduction of the striatal Bcl-2 levels may be the disruptive effect of SD on the cytoskeletal proteins finally leading to the apoptotic cell death.et al. showed that incubation of the hippocampal neurons in the BDNF-enriched media for 24 hr increases Bcl-2 expression level  protein (et al. showed, SD prevents Akt phosphorylation through reduction of Ca2+ influx, which ultimately leads to the elevation of pro-apoptotic proteins and their subsequent apoptogenic activities (et al. reported that reduction in the locomotor activity of the Parkinsonian rats is averted by the induction of SD for 22 consecutive days. Also, their findings showed that SD not only elevates DA levels but also increases its turnover (An increase in the striatal Bax protein following SD in the Parkinsonian rats can be attributed to the dysregulation of PI3K/AKT pathway. Akt phosphorylation by Ca protein . BAD fac protein . However protein . Then, B protein . As a sttivities . In our tivities . This returnover . Neuroimturnover . The actturnover . This nuturnover . Therefoturnover , 67. It turnover . Neverthturnover , which cturnover . TherefoSD suppressed the anti-apoptotic compensatory responses in the striatum. Therefore, it may accelerate continual neuronal cell death in PD. Given the fact that sleep disorders are frequently experienced by PD patients, management of this complication may slow down progressive DAergic neuronal loss. Due to complex neuronal effects of SD, further preclinical and clinical studies should be designed to find out its exact role in PD.


Prior to mass spectrometry, on-line sample preparation can be beneficial to reduce manual steps, increase speed, and enable analysis of limited sample amounts. For example, bottom-up proteomics sample preparation and analysis can be accelerated by digesting proteins to peptides in an on-line enzyme reactor. We here focus on low-backpressure 100 μm inner diameter (ID) × 160 mm, 180 μm ID × 110 mm or 250 μm ID × 140 mm vinyl azlactone-co-ethylene dimethacrylate [poly(VDM-co-EDMA)] monoliths as supports for immobilizing of additional molecules , as the monolith was expected to have few unspecific interactions. For on-line protein digestion, monolith supports immobilized with trypsin enzyme were found to be suited, featuring the expected characteristics of the material, i.e., low backpressure and low carry-over. Serving as a functionalized sample loop, the monolith units were very simple to connect on-line with liquid chromatography. However, for on-line target deconvolution, the monolithic support immobilized with a Wnt pathway inhibitor was associated with numerous secondary interactions when exploring the possibility of selectively trapping target proteins by drug-target interactions. Our initial observations suggest that (poly(VDM-co-EDMA)) monoliths are promising for e.g., on-line bottom-up proteomics, but not a “fit-for-all” material. We also discuss issues related to the repeatability of monolith-preparations. With the recent advances in liquid chromatography (LC) and mass spectrometry (MS), sample preparation is the most time-consuming part of the method and is often the largest contributor to false analysis results. Sample preparation may be separated into two main categories: off-line procedures and on-line procedures. In on-line sample preparation techniques, the samples are prepared and measured in the same workflow in a closed system, often offering improved performance as both loss of sample and possibility of contamination is reduced. In an automated on-line method the contribution from human error is reduced, increasing the repeatability/reproducibility of the method Kataoka, .A specific on-line sample preparation step that can be beneficial to have up-stream in an LC-MS analysis system, is the digestion of proteins to peptides for bottom-up proteomics. This can be achieved by immobilizing enzymes on suitable supports compatible with a capillary- or nanoLC-MS set-up, e.g., particle packed , dense packing and narrow pores (10 to 30 nm/100 to 300 Å). The high pressure force the liquid to flow around the particles instead of into the pores, reducing the surface of active sites  available to interact with the proteins  reactor (“monolith trapped drug”), as a possible tool for target deconvolution in drug discovery. The CRAM reactor would be used to trap the drug target through drug-target interactions, and subsequently elute purified target eluate for identification. The Wnt-inhibitor anti-cancer drug (LDW639) targeting tankyrase 1 and 2 (TNKS1/2) in the Wnt/β-catenin signaling pathway was selected as the model system for the CRAM reactor  as initiator. In brief, the fused silica capillaries were filled with 1 M NaOH using an previously described in-house pressurized filling system and sealed in both ends by septa . The NaOH treated capillaries were filled with a silanization solution , which was sonicated for 5 min before filling. The filled capillaries were sealed by septa and placed in an oven  at 110°C for 6 h. Subsequently, the capillaries were flushed with acetonitrile (ACN) and dried with N2(g). The silanization procedure was based on Hustoft et al. (2(g). The polymerization procedure was based on Geiser et al. (1H-NMR), further details are given in The overview of chemicals used in the following experiments are presented in t et al. . Finallyr et al. . The cheFor characterization of the morphology of the monoliths, a micrograph of the cross-section was captured with a Quanta 200 FEG-E scanning electron microscope (SEM) from FEI Company  now a part of Thermo Fisher Scientific. From the dry poly(VDM-co-EDMA) monolith, 1 cm was cut off and glued in an upright position on a sample holder with carbon tape. The sample holder was placed in the sample chamber before the chamber was pumped to low vacuum. A large field detector operating at 15.0 kV, 12 mm distance for the sample and with a 4.0 spot size was used to capture the micrographs.For immobilizing trypsin to VDM on the monolithic support, a solution consisting of 0.25 mg/mL trypsin and 2.25 mg/mL benzamidine in 50 mM phosphate buffer (pH 7.2) was flushed through the monoliths for 3.5 h. The resulting immobilized enzyme reactors (IMERs) were filled with 50 mM ammonium acetate buffer (pH 8.75), sealed with septa and stored at 4°C.I, beta-keto ester, 95%) and 4-boc-aminomethylbenzamidine , and modified by the addition of a linker  all purchased from Fluorochem . The finalized product (VII), structure shown in VII) is described in The modified LDW639 Wnt-inhibitor drug was synthesized in-house from methyl-4-oxotetrahydro-2H-thiopyran-3-carboxylate (The CRAM reactor was prepared by immobilizing the modified LDW639 Wnt-inhibitor drug on the poly(VDM-co-EDMA) monolith by flushing a solution of 5 mg/mL drug in 50 mM phosphate buffer (pH 7.2) through the capillary for 3 h (~0.5 mL). Additionally, a reference monolith was made by flushing 1 M monoethanolamine (MEA) through a monolith in the same manner. Both the CRAM reactor and the MEA monolith were filled with 50 mM phosphate buffer, sealed with septa and stored at 4°C.For evaluation of the protein digestion potential of the immobilized enzyme reactors, solutions of 500 μg/mL reduced [by dithiothreitiol (DDT)] and alkylated [by iodoacetamide (IAM)] myoglobin dissolved in 50 mM ammonium acetate were used. The in-solution digested myoglobin solutions were prepared by adding 10 μg trypsin per 500 μg myoglobin, and incubation at 37°C for 45 min. The solutions were stored at −20°C.To investigate the possibility of trapping the protein target of the immobilized Wnt-inhibitor, human embryonic kidney 293 (HEK293) cells were received, after cell cultivation, from the unit of Cell Signaling, Oslo University Hospital. The cell line HEK293 were purchased from American Type Culture Collection , and maintained according to the ATCC guidelines. For cell lysis with a non-denaturing buffer , diluted with water to 40 mL), a procedure based on Voronkov et al.  was used−1. A gradient was run at a flow rate of 10 μLmin−1 for 25 min bypassing the IMER (started after 1 min); at 0% B for 0–1.5 min, linearly increased to 55% B for from 1.5 to 17 min, kept at 55% B at 17–23 min, quickly increased to 90% B for 1 min and then reversed to 0% B for 1 min. Detection was performed at 210 nm by a WellChrome K-2600 UV-detector , equipped with a 65 nL (100 μm ID/375 μm OD) flow cell. A Perkin Elmer Nelson 900 series analog-to-digital interface  and a computer with a TotalChrom software were used for obtaining the chromatograms.For evaluation of protein digestion by the IMERs, an on-line IMER-LC-UV system utilizing an Agilent 1,100 series pump   and collected in a tube marked “EluateF,” which was added 5 μL of 1 M NaOH prior to and 5 μL following sample collection to immediately neutralize the sample. A second elution was performed in the same manner with 2% sodium dodecyl sulfate  in 60 mM tris buffer (w/v) and collected in a tube marked “EluateS.” All of the collected eluates were analyzed by western blot for TNKS1/2 using actin as loading control. Western blot procedure is explained in detail in An EASY nLC pump from Proxeon now a part of Thermo Fisher  monolith was assessed as a support for immobilized ligands to enable on-line sample preparations in bottom-up proteomics and target deconvolution in drug discovery using trypsin and modified LDW639, respectively.−14 m2, calculated as described in Meller et al.  monolith was suitable as a support for immobilized trypsin to enable fast on-line digestion of proteins to peptides. An evaluation of 9 replicates of 100 μm ID × 160 mm and 9 replicates of 180 μm ID × 110 mm poly(VDM-co-EDMA) monoliths were successfully prepared with a homogeneous, uniform morphology, shown in r et al. , based or et al. , commercr et al. , and silr et al. . The perr et al.  to that r et al. . The pepr et al. ; proteinr et al. . HoweverThus, the poly(VDM-co-EDMA) based trypsin IMERs allow easy manual injection due to low backpressure  and a simple washing step eliminates possible carry-over after successful digestion of myoglobin in 5 min. The trypsin poly(VDM-co-EDMA) IMERs can be used up-stream LC-MS in bottom-up proteomic studies to reduce time consumption on sample preparation and loss of sample.In drug discovery, development and optimization of new (and old) drugs depends on target identification  monoliths immobilized with modified LDW639 drug for trapping and purification of target tankyrase 1/2 was executed. The modified LDW639 drug was successfully synthesized and found to inhibit Wnt-signaling in STF-Luc assay . Both thThe eluates collected from the CRAM reactor and the MEA monolith after applying HEK293 cell lysate were analyzed by western blot for target TNKS1/2 using actin as loading control, because actin is commonly expressed in all eukaryotic cell types. TNKS1/2 was not found in any eluates collected during flushing, washing or eluting from the CRAM reactor or the MEA monolith . TNKS1/2The CRAM reactor used in this study shows that common elution conditions in affinity purification is not sufficient for eluting trapped target proteins from immobilized drugs. However, elution with 2% FA was sufficient for elution of peptides from poly(VDM-co-EDMA) monoliths with immobilized antibodies  monolith in 180 μm ID capillaries, the monolith was also prepared in 250 μm ID capillaries. At this stage of the project, the uniform morphology was only successful in 250 μm ID capillaries . In contcompared . VirtualHence, we could not trace repeatability issues to the chemicals employed, suggesting that other conditions/factors e.g., subtle variations in humidity and temperature may play more substantial roles for the morphology of the monoliths . One of the reviewers suggested that including a protonation step of the silanols, using HCl after treatment of NaOH, may give a better activation of the silica surface before silanization by γ-MAPS and consequently a more consistent attachment of the monolith. Concerning the polymerization, the solution of the monomer EDMA consisted of 90–110 ppm of monomethyl ether hydroquinone . The MeQH has been suggested to be removed prior to polymerization to increase reproducible morphology of the monoliths.The poly(VDM-co-EDMA) monolith has a good mechanical strength suitable for attachment as a loop on a 6-port valve and is easily prepared in fused silica capillaries with various IDs. The reactive VDM monomer also allows for tailoring toward specific applications by post-modification with ligands after formation of the organic polymer.The poly(VDM-co-EDMA) monoliths shows potential as trypsin-based IMERs with low backpressure and fast digestion of protein standards, and was very simple to incorporate in an on-line system as a functionalized loop with little carry-over. A next step will be to investigate their potential in on-line systems to be used in e.g., the field of “organ-on-a-chip” mass spectrometry-based proteomics. We have undertaken initial steps toward on-line drug/target studies using monolithic supports. Based on the same type of poly(VDM-co-EDMA) monolith, the CRAM reactor revealed that selective trapping and subsequent purified elution of high molecular weight protein targets (>110 kDa) in complex cell lysate was not straightforward, highlighting some limitations of this otherwise promising material and format. Nonetheless, due to the advantages that on-line drug/target studies can have, we are encouraged to explore alternative monoliths due to the format's versatility and ease of coupling with analytical instrumentation.Datasets generated for this study are included in the article/CO, FS, and OB contributed to monolith work. CO performed LC-UV and LC-MS experiments. CO and FS performed biochemical experiments. CO, PR, and CS performed drug synthesis. SW, EL, and PR were supervisors. All authors contributed to planning experiments and writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Wirelessly powered, very compact sensors are highly attractive for many emerging Internet-of-things (IoT) applications; they eliminate the need for on-board short-life and bulky batteries. In this study, two electrically small rectenna-based wirelessly powered light and temperature sensors were developed that operate at 915 MHz in the 902–928-MHz industrial, scientific, and medical (ISM) bands. First, a metamaterial-inspired near-field resonant parasitic (NFRP) Egyptian axe dipole (EAD) antenna was seamlessly integrated with a highly efficient sensor-augmented rectifier without any matching network. It was electrically small and very thin, and its omnidirectional property was ideal for capturing incident AC wireless power from any azimuthal direction and converting it into DC power. Both a photocell as the light sensor and a thermistor as the temperature sensor were demonstrated. The resistive properties of the photocell and thermistor changed the rectifier’s output voltage level; an acoustic alarm was activated once a threshold value was attained. Second, an electrically small, low-profile NFRP Huygens antenna was similarly integrated with the same light- and temperature-sensor-augmented rectifiers. Their unidirectional nature was very suitable for surface-mounted wireless power transfer (WPT) applications . Measurements of the prototypes of both the light- and temperature-sensor-augmented omni- and unidirectional rectenna systems confirmed their predicted performance characteristics. Wirelessly powered sensors are very attractive for many emerging Internet-of-things (IoT) applications, especially since the number of wireless IoT devices is expected to experience exponential growth in the upcoming 5G era ,2,3,4,5.ka < 1, where k is the wave number at the operating frequency and a is the radius of the smallest sphere enclosing the entire rectenna), large wireless power capture capacities, and high AC-to-DC conversion efficiencies simultaneously.Different wireless IoT application scenarios would require sensors powered by rectennas with a diverse set of wireless power capture characteristics. For example, a light, temperature, or humidity sensor could be placed on a tunnel’s ceiling and powered by radio frequency (RF) sources stationed at points along it. As illustrated in ka value being 1.66. The peak realized gain is 1.8 dBi and the maximum AC-to-DC conversion efficiency is only 75%. In comparison, the size of the linearly polarized omnidirectional rectenna reported herein is much smaller: 49.4 × 49.4 mm; hence, it is electrically small, with its ka being only 0.47. Even though its receive area is six times smaller in comparison, the realized gain is 1.75 dBi and the maximum AC-to-DC conversion efficiency is 87%. The temperature and light sensors are organically combined with these omni- and unidirectional rectennas to achieve very compact systems. In this article, temperature (thermistor) and light (photodiode) sensors that operate at 915 MHz in the 902–928-MHz industrial, scientific, and medical (ISM) bands are presented that are powered by both electrically small, highly efficient, metamaterial-inspired omnidirectional and Huygens dipole  rectennas. These rectennas were designed for their maximum WPT conversion efficiency. They have several advantages over previously reported systems. For example, the linearly polarized omnidirectional rectenna reported in [λ0) and very thin (thickness is 0.002 λ0) with a ka value less than 0.5 (ka = 0.47). The highest gain of the EAD antenna is 1.75 dBi. The wireless power capture capacity of this EAD system is similar to a classic half-wavelength dipole rectenna, but its diameter is more than 3.8 times smaller. The simulated and measured characteristics of its optimized prototype are in good agreement.First, a linearly polarized (LP) near-field resonant parasitic (NFRP) omnidirectional Egyptian axe dipole (EAD) rectenna was integrated with both a temperature- and a light-sensor-augmented rectifier circuit. It is a more advanced version of the previously reported NFRP rectenna in . The entka ≈ 0.73), is low profile (~0.04 λ0), has a large wireless power capture capacity (antenna gain is 3.8 dBi), and attains a very high (87.8%) conversion efficiency.Second, an advanced version of the electrically small Huygens dipole antenna concept  was simiThe performance characteristics of both the photocell-enabled sensors for light detection and the thermistor-enabled sensors for temperature assessment are discussed. The output DC voltage of the rectifier is controlled by the impedances of these sensors, which vary according to the conditions they are sensing. An acoustic device is connected to the output of the rectifiers as a threshold alarm for both the light and temperature levels. When the environment is too bright or the temperature is too high, the alarm will be activated. The developed highly compact wirelessly powered sensor systems are ideal candidates for quality control, security and hazard detection, and health monitoring applications, all of which are natural prospects for emerging wireless IoT systems. Electrically small, omnidirectional WPT-driven light and temperature sensors have been developed. They can monitor light conditions or ambient temperatures. These WPT-driven sensors consist of two primary parts: a receiving EAD antenna and a sensor-augmented rectifier circuit. Measured results from an optimized prototype validated their simulated performance characteristics.EADR = 24.7; straightW = 2.0; armW = 3.0; armL = 14.6; EADG = 9.0; drivenL = 44.4; drivenW = 3.0; drivenG = 35.2; L1= 5.0; and G1= 1.5. The entire system was highly compact (ka = 0.47) and easy to fabricate.An electrically small EAD antenna with an inductive input impedance was designed to be directly matched to a highly capacitive rectifier circuit without any matching elements at 915 MHz in the corresponding ISM band and is illustrated in The EAD receiving antenna alone was simulated with the ANSYS Electromagnetics Suite 16.2. The input impedance and realized gain patterns are shown in C1 and C2 facilitated impedance matching. C2 also acted as an energy storage component during the negative portion of the sinusoidal AC signal. Two HSMS286C Schottky diodes, D1 and D2, were arranged to yield a voltage doubler. C3 acted as a low-pass filter and smoothed the output DC voltage. The sensor itself can be any impedance-dependent element, such as a photocell or thermistor. LR was the load resistor. cL1 and cL2 were the RF chokes. The detailed values of these components were: C1 = 0.4 pF; C2 = C3 = 100 pF; and cL1 = cL2 = 560 nH. The LR = 3.6 kΩ for light detection and 560 Ω for temperature sensing. The circuit model of the sensor-augmented rectifier is shown in The light detection was enabled with a photocell from Adafruit™  that hadtG = 7.0 dBi at 915 MHz (λ0 = 327.64 mm). The distance R between the horn antenna and the rectenna was 1.2 m, which was in the far-field region of the horn according to the standard criterion 2D2/λ0 [rP was then determined according to the selected output power from the signal generator which defined tP. tP represents the power transmitted from the horn antenna. It was the net result of the output power of the signal generator, the gain of the power amplifier, and the losses incurred in the cables. The optimized sensor-augmented rectenna was fabricated, assembled, and tested. The measurement setup was built in an anechoic chamber, as shown in n 2D2/λ0 . The gaiThree sets of experiments were conducted. First, the output DC voltage and the AC-to-DC conversion efficiency of the rectenna with the 5.1-kΩ resistor load (without the sensor) were measured. Second, the output DC voltage values of the rectenna with the photocell present for different brightness levels were measured. An acoustic alarm from PUI Audio™  was attaφ = 0°, θ = 180°). The peak measured efficiency was 89.9%, which was very close to its simulated value, 87.8%.In the first set of experiments, the output DC voltage as a function of the source frequency and the AC-to-DC conversion efficiency as a function of the received power were obtained. These results are shown in y-axis, as shown in φ = 0°, θ = 180°), 6.7 V , 6.4 V , and 6.7 V , respectively. Very good omnidirectional wireless power capture capacity was observed.To evaluate its omnidirectional wireless power capture capacity, the EAD rectenna was rotated at 45° steps around the In the second set of measurements, the photocell augmented the rectifier. The load was replaced by the 3.6-kΩ resistor. The received power was fixed at 9.5 dBm. As shown in In the third set of the measurements, the thermistor replaced the photocell and the load resistance was changed to 560 Ω. Unfortunately, it was difficult to control the exact temperature in the anechoic chamber. Consequently, an alternative was adopted to mimic the resistance of the thermistor at different temperatures. Recall that the resistance of the thermistor as a function of the temperature is shown in Electrically small, light- and temperature-sensor-augmented Huygens rectennas have been developed. They are suitable, for example, for mounting on walls in a room for unidirectional power capture. As with their EAD counterparts, they can monitor light conditions or ambient temperatures. The Huygens rectenna design is based on those reported previously ,36. HoweEADR = 36.8; straightW = 7.0; armW = 4.0; EADG = 29.8; CLLR = 30.4; CLLL = 58.5; CLLH = 12.77; CLLW = 6.0; CLLG = 5.0; drivenL = 15.56; drivenW = 0.7; and FoamH = 7.5. The sensor-augmented rectifier was the same as that used in the EAD rectenna.The electrically small Huygens antenna design is shown in As was achieved with the EAD rectenna, the Huygens rectenna was also designed to have an inductive impedance for direct matching to the rectifier. This was realized simply by adjusting the length of the dipole element. As shown in ka = 0.73) and of low profile (H = 0.04 λ0). The TDK™ thermistor with its SMD (Surface Mount Device) package was integrated into the rectifier, as shown in The optimized sensor-augmented Huygens rectenna was fabricated, assembled, and tested. The prototype is shown in The output DC voltage values as functions of the source frequency and the AC-to-DC efficiency values as functions of the received power were measured in the first experiment. The results are shown in The performance of the Huygens rectenna augmented with the photocell and the thermistor were also evaluated. The measured results are shown in This article reported the investigations of both electrically small, highly compact sensor-augmented omnidirectional and Huygens  dipole rectennas for wirelessly powered IoT sensing applications. Both the EAD and Huygens rectennas performed as predicted. The experimental results demonstrated that both rectennas achieved high AC-to-DC conversion efficiencies . The EAD rectenna exhibited a large omnidirectional wireless power capture range. While the Huygens rectenna had a smaller  capture range, it had almost double the capture capability in that range . The PCB fabrication of both rectenna systems is straightforward and expedites their minimal cost levels. The EAD rectenna costs less than the Huygens rectenna simply because it only requires one PCB substrate. In both cases, the predicted performance characteristics of the light (photocell) and temperature (thermistor) sensor-augmented versions were successfully confirmed. The measured results demonstrated their effective light and temperature detection and the simultaneous ability for them to drive an alarm once a threshold voltage was exceeded. These wirelessly powered sensor systems illustrate the society-impactful potential for many emerging IoT wireless sensor-based applications.


The chemical structure and modified surface were characterized by FT-IR, 1H NMR, X-ray photoelectron spectroscopy (XPS), and water contact angle. The SPEU-PEGs displaying much lower water contact angles (23.9–21.8°) than SPEU (80.5°) indicated that the hydrophilic PEG chains improved the surface hydrophilicity significantly. The SPEU-PEG films possessed outstanding mechanical properties with strain at break of 866–884% and ultimate stress of 35.5–36.4 MPa, which were slightly lower than those of parent film, verifying that the chemical treatments had minimum deterioration on the mechanical properties of the substrate. The bovine serum albumin adsorption and platelet adhesion tests revealed that SPEU-PEGs had improved resistance to protein adsorption (3.02–2.78 μg/cm2) and possessed good resistance to platelet adhesion (781–697 per mm2), indicating good surface hemocompatibility. In addition, due to the high grafting density, the molecular weight of surface-grafted PEG had marginal effect on the surface hydrophilicity and hemocompatibility.In the paper, poly(ethylene glycol) (PEG) was grafted on the surface of poly(ester-urethane) (SPEU) film with high grafting density for biomedical purposes. The PEG-surface-grafted SPEU (SPEU-PEG) was prepared by a three-step chemical treatment under mild-reaction conditions. Firstly, the SPEU film surface was treated with 1,6-hexanediisocyanate to introduce -NCO groups on the surface with high density (5.28 × 10 Segmented polyurethane (SPU) is widely used in biomedical fields, such as cardiovascular devices, artificial organs, and tissue engineering scaffolds, due to its long-term bio-stability, excellent mechanical properties, and relatively superior biocompatibility ,3,4,5,6.Many kinds of surface modification approaches, including chemical treatment, strong oxidation, plasma treatment, UV irradiation, and laser treatment have been used to modify the surface of medical SPU ,18,19,20−7 mol/cm2, and the results of protein adsorption and platelet adhesion tests showed that the surface hemocompatibility needed to be further improved. In consideration of there being four urethane groups in each hard segment, the SPEU possesses higher −NH- content than MDI-based PU, and thus, the high grafting density on the film surface could be obtained by chemical treatment with diisocyanate.In our previous report , a new b2 groups of tris(2-aminoethyl)amine (TAEA) via condensation reaction (2 on the SPEU surface (SPEU-NH2); finally, the PEG was grafted on the SPEU (SPEU-PEG) surface through Michael addition (1H NMR), X-ray photoelectron spectroscopy (XPS), and water contact angle measurements. The influence of chemical treatments on the mechanical properties of the films was researched, and surface hemocompatibility of the PEG-grafted SPEU films was evaluated by protein adsorption and platelet adhesion tests. Furthermore, the effect of molecular weight of grafted PEG on the surface hydrophilicity and hemocompatibility was preliminarily studied.In this study, we graft PEG on the surface of SPEU film to obtain a high grafting density without deteriorating the intrinsic mechanical properties of the substrates. The surface grafting of PEG on SPEU films was prepared by a three-step chemical treatment , which wreaction b to immoaddition c betweenMn = 2000 g/mol) was supplied by Shenzhen Polymtek Biomaterial Co., Ltd.  and dried for 4 h at 100 °C under vacuum prior to use. HBH was synthesized in our lab according our published paper [1H NMR, 13C NMR, and HR-MS. HDI and dibutyltin dilaurate (DBTDL) were purchased from Sigma-Aldrich Chemical Co.  and used without further purification. TAEA,  was dried over 4-Å molecular sieves and redistilled before use. APEG  was obtained from Shanghai Aladdin Reagent Co.  and was used as received. N,N-dimethylformamide  was dried with phosphorus pentoxide and distilled under reduced pressure before use. Other reagents were AR grade and purified by standard methods.Poly(ε-caprolactone)  in the FT-IR spectrum disappeared (~3.5 h), and subsequently diluted with DMF to approximately 4.5 g/100 mL. The diluted solution was poured into a Teflon mold. The solvent was removed by natural volatilizing at 50 °C for 4 days, and the semitransparent films with 0.20 ± 0.02 mm thickness were subsequently vacuum dried at 40 °C for 1 day to remove the last traces of solvent. GPC (THF): Mw = 111000, Mn = 83000, Mw/Mn = 1.33.SPEU was synthesized according to our previous publication . Briefly2 film. Finally, the PEU-NH2 film was immersed in 10 mL absolute ethanol containing APEG with different molecular weight (0.3 mmol/mL). After gentle shaking for 12 h at room temperature, the films were rinsed with absolute ethanol thoroughly and dried under vacuum at room temperature to constant weight. Three SPEU-PEGs based on APEG with Mn = 1200, 2400 and 4000 g/mol were named as SPEU-PEG-a, SPEU-PEG-b, and SPEU-PEG-c, respectively.Before chemical treatments, the surfaces of the SPEU film discs with ~10 mm diameter were ultrasonically cleaned in 50% ethanol for 30 min and then dried in a vacuum. First, HDI (1.0 g) and DBTDL (0.02 g) were dissolved in anhydrous toluene (10 mL) to get a homogeneous solution, SPEU disc was immersed in the solution and the reaction was allowed to shake for 3 h at room temperature. The film disc was taken out and rinsed with anhydrous toluene to remove unreacted HDI from the surface completely. The SPEU-NCO film achieved was then soaked in 10 mL anhydrous toluene containing 0.13 g TAEA. After gentle shaking at 18 °C for 6 h, the film disc was removed from the solution and flushed with anhydrous toluene to obtain the PEU-NH−1 with the resolution of 4 cm−1.FT-IR: FT-IR spectra were recorder on an Alpha infrared spectrometer  equipped with a Bruker platinum ATR accessory in the range of 4000-400 cm1H NMR: NMR spectra were recorded with a 400 MHz Avance II spectrometer  with CDCl3 as the solvent.Mn) and molecular weight distribution (w/MMn) were measured by gel permeation chromatography . The continuous phase was tetrahydrofuran, and monodisperse polystyrene was used as the calibration standards.GPC: The number average molecular weight  at room temperature. Dumbbell-shaped specimens were punched from the films with a punching die of 12 mm width and 75 mm length, the neck width and length were 4.0 and 30 mm, respectively. The crosshead speed was controlled at 50 mm/min. At least five specimens were measured and the averaged results were reported.−7 Pa. The binding energies were referenced to the C1s line at 284.8 eV from adventitious carbon. The measurement was carried out at room temperature with a 90° take-off angle of the photoelectron. The atomic concentrations of the elements were determined by their corresponding peak areas.XPS: Chemical composition of the film surface was characterized by XPS  with a mono-chromated Al-Kα radiation source (energy 1486.68 eV). The base pressure for the measurement was ~3 × 10Water contact angle: The static water-contact angles of blank and modified SPEU films were measured on a drop shape analysis system  at room temperature. Ultrapure water was used as test fluid, and the contact angles were read within 3 s after each drop by using a microscopy. At least six replicate measurements were performed on each specimen and the average was calculatedProtein adsorption: Bradford’s protein determining method was used to assay the albumin adsorbed on the film surface with bovine serum albumin (BSA) as the model protein ,27. Befov:v) with a step of 10% for 30 min in each step. Finally, the platelet-attached surfaces were allowed to dry at room temperature and coated with gold prior to being observed with a Cold Field Emission Scanning Electron Microscope . Different fields were randomly observed. Quantification of adhered platelets was calculated from eight different areas of one specimen using FE-SEM images.Platelet adhesion: The interaction between the film surface and blood was evaluated by platelet adhesion tests. The platelet-rich plasma (PRP) was prepared from fresh rabbit blood containing sodium citrate as an anticoagulant  according the reported literature . The fil−7 mol/cm2, which was much higher than that of MDI-based PU  in the reported paper in Reference [−3 mol/g, while the -NH- content in Pellethane® PU was only 0.9 × 10−3 mol/g (the result is based on the same soft segment content in SPEU with Pellethane® PU). After introducing the -NCO groups onto the SPEU surface via allophanate reaction with HDI, a sharp peak at 2270 cm−1, the characteristic symmetric stretching vibration peak of -NCO, was observed in the FT-IR spectrum of SPEU-NCO film , the spectra of SPEU-PEG-a are shown in 2 absorption peak at 1611 cm−1 disappeared thoroughly, and the peak intensity at 3329 cm−1 increased slightly which was due to the reaction between -NH2 group and APEG. In addition, the characteristic absorption bands at 1166 cm−1 and 1095 cm−1 belonged to the stretching vibration of ester bonds C-O-C of SPEU and ether bonds C-O-C of grafted PEG. In the 1H NMR spectrum, the proton signals of double bond at δ 5.25 and δ 5.92 ppm  are illustrated in 2 film increased obviously with the content increasing to 10.3%, which indicated that -NH2 groups had been immobilized onto the surface. Compared to SPEU-NH2, the nitrogen content of SPEU-PEG decreased significantly, and with the increasing molecular weight of surface-grafted PEG, the nitrogen content decreased gradually. After the PEG is grafted on the surface with high grafting density, the PEG chains can form a thin layer coating on the surface which hinders the inner nitrogen element to be detected, leading to the low surface nitrogen content. The results confirmed that PEG was successfully immobilized onto SPEU surface.The presented XPS investigations were performed in order to extract quantitative results about the surface composition. The overview XPS spectra of the SPEU, SPEU-NH2, SPEU-PEG-a, SPEU-PEG-b, and SPEU-PEG-c films displayed analogous stress-strain behaviors with the strain at break of 866–884% and ultimate stress of 35.5–36.4 MPa  after the ultrapure water is dripped onto the film surface, otherwise the water drop will spread out on the surface. The PEG grafted on the surface can interact with water molecules by the role of hydrogen bonding to form hydration layer on the film surface, resulting in low water contact angle. In addition, SPEU-PEGs exhibiting similar water contact angle indicated that the molecular weight of grafted PEG slightly affected the surface hydrophilicity, which should be due to the high grafting density of PEG on surface. These results supported that PEG had been grafted onto the SPEU surface and the hydrophilicity of PEG-grafted SPEU was improved significantly.The surface hydrophilicity of the blank and modified SPEU films were characterized by sessile contact angle measurement, and the results are presented in 2; SPEU-PEG-b: 2.88 μg/cm2; SPEU-PEG-c: 2.78 μg/cm2) was significantly lower than that of blank surface (SPEU: 11.87 μg/cm2), which was ascribed to the surface-grafted PEG. PEG chain not only has excellent hydrophilicity, but also possesses high flexibility. The highly flexible and well-hydrated chains can affect the fluidity of blood and hinder the protein adsorption on the surface [Protein adsorption on the surface of a material is always considered as the first step to evaluate the blood compatibility of the implanted or blood-contact biomaterials . Figure  surface . The SPE surface , which sWhen blood is exposed to a foreign surface, initial protein adsorption on the surface is followed by platelet adhesion and release, which brings about cellular thrombogenesis . Thus, p1H NMR, XPS, and water contact angle. The SPEU-PEGs displaying a much lower water contact angle than SPEU indicated that the hydrophilic PEG chains improved the surface hydrophilicity significantly. The mechanical properties of the SPEU-PEG films were slightly lower than that of parent film, verifying that the chemical treatments had minimum deterioration on the mechanical properties of the substrate. The low BSA adsorption quantity (2.78–3.02 μg/cm2) and good anti-platelet adhesion capacity (781–697 per mm2) revealed that SPEU-PEGs had improved surface hemocompatibility. Moreover, due to the high grafting density, the molecular weight of grafted PEG had marginal effect on the surface hydrophilicity and hemocompatibility. The PEG-grafted SPEU films possessed outstanding mechanical properties  and good surface hemocompatibility, implying its high potential to be applied as long-term implants and blood-contacting biomaterials.In the paper, PEG was grafted on the surface of SPEU film with high grafting density to improve surface hemocompatibility. The PEG-grafted SPEU (SPEU-PEG) was prepared by three-step chemical treatments  under mild reaction conditions. The surfaces were characterized by FT-IR,


The HW-group had 70% more fat mass at 5 mo, and then showed marked catch-down in BAZ from 5 to 18 mo (−0.84). Milk intake at 5–6 mo did not differ between the groups. In the HW-group milk-leptin was lower at 5 mo and serum-leptin was considerably higher at 5 and 9 mo compared to the NW-group. Serum-leptin at 5 mo was positively associated with weight-for-age z-score (WAZ) and fat mass and negatively with WAZ change from 5 to 9 mo. In conclusion, breastfed infants with EWG had catch-down growth when other foods were introduced. Low milk-leptin in the HW-group may have stimulated appetite and milk intake when weight gain was high. High serum-leptin in the HW-group suggests early leptin resistance, which could impact cerebral regulation of energy intake. Larger studies are needed to confirm these results.Some infants experience excessive weight gain (EWG) during exclusive breastfeeding, but causes and consequences are unknown. The objective was to identify factors associated with early EWG. Infants with EWG (HW-group) were examined at 5, 9 and 18 mo and compared to a breastfed group with normal weight gain (NW-group). Anthropometry, body composition, milk and blood samples, and milk intake were measured. Mean body-mass-index-for-age  Breastfed infants have a growth pattern different from formula fed infants. They grow more rapidly during the first 1–2 months (mo) and then more slowly—both weight gain and linear growth—in the first years . There aSome infants experience excessive weight gain (EWG) during the period they are exclusively breastfed (EBF) and have a marked catch-down when complementary foods are introduced, suggesting that the EWG is caused by breastfeeding ,9,10. StThere is convincing evidence from several meta-analyses that a high weight gain during infancy, especially the first six months, is associated with an increased risk of overweight, obesity, and metabolic complications later in life ,13,14,15Although the case reports suggest that at least some breastfed infants with EWG show catch-down growth when other foods are introduced, it is important to examine if these infants are at risk of later obesity and metabolic complications. A small exploratory cohort of infants with EWG during EBF was established in order to identify predictors and consequences of the EWG and a cohort with normal weight gain was recruited for comparison. Participants were part of an ongoing prospective observational cohort study, the SKOT III cohort, consisting of two groups; a high weight gain group (HW-group) and a normal weight gain group (NW-group).z-score (WAZ) >2.00. The infants were qualified for enrollment to the HW-group if they had an increment of at least +1 SDS in WAZ within the first 5 mo postpartum. The NW-group was recruited through the National Civil Registry. Families from the Copenhagen area (n = 805) with infants born between 15 July 2016 and 31 August 2016 were invited by postal invitation to participate. Inclusion criteria for the NW-group were infants aged 4–6 mo with a WAZ between −1.0 and +1.0 SDS. Other inclusion criteria were similar for both groups: exclusively or fully breastfed  for at least 4 mo. At first visit the infants were to be 5–6½ mo old and have breastfeeding as the primary energy source not receiving more than two meals of solid food per day. Both HW- and NW-group mother-infant dyads were examined at infant’s age 5–6½ mo and 9 mo ± 2 weeks, and the HW-group further at 18 mo ± 4 weeks.EBF infants considered to have excessively high weight-for-age were referred via Danish health workers who had been informed about the study through postal letters and social media from August 2015 to October 2016. High weight was defined as infants aged 4–6 mo having a weight-for-age The study protocol was approved by the Regional Ethical Committee of the Capital Region of Denmark in accordance with the Helsinki declaration (H-15008948) and the Data Protection Agency (2015-57-0117 & 2015-57-0116) and written informed consent was obtained from parents.All anthropometric measurements at the three visits were carried out at the Department of Nutrition, Exercise and Sports, University of Copenhagen, Denmark. Weight and length from birth to 5 mo measured by health nurses and general practitioners, were reported by the parents and used for assessing recruitment criteria.z-scores; WAZ, length-for-age z-score (LAZ), BAZ, triceps skinfold-for-age (TSFZ) and subscapular skinfold-for-age (SSFZ) were obtained using the software WHO Anthro [The weight of naked infants was measured using a digital pediatric scale giving an average of 40 measurements recorded to the nearest gram . The following anthropometric measurements were performed in triplicate and the average was used: recumbent length to the nearest 0.1 cm , mid-upper-arm, head, lower leg, recumbent waist and thorax circumferences to the nearest millimeter , and triceps and subscapular skinfold thickness to the nearest 0.1 mm . Sex and age specific O Anthro .Mothers’ pre-pregnancy weight, pregnancy weight gain, and the fathers’ weight and height were self-reported, while the mothers’ weight and height were measured at all three visits .Body composition was measured using a tetrapolar Bioelectrical Impedance Analyzer (BIA) Quantum III  which uses single frequency (50 kHz) between right hand and right foot to measure whole body resistance. Prediction of fat free mass (FFM) for each infant was completed using an equation developed by Lingwood et al. . DetailsMothers were instructed to use an electronic baby weighing scale  for weighing their infants to measure the 24 h milk intake at 5 mo. For a period of 72 h mothers weighed their infants before and after each breastfeeding-session (each feed) without change in clothing or diaper. Calculation of intake in grams was done by subtracting weight of the infant before the feed from the weight after the feed. In cases where test weighing was not completed for all feeds, the intake was estimated using an average of intake per feed calculated from the mother’s registration. The first feed after the 72 h period was used as the end point for registration period, and in cases where these data were missing it was estimated from the three previous days. A corrected 24 h milk intake for each infant was determined by dividing the intervals in hours and multiplying by 24. No correction for infant insensible water loss was made, and therefore the milk intake is likely to be underestimated by 3–10% ,23. FeedAt the 5 and 9 mo visits the mothers were asked to collect 10 mL foremilk and 10 mL hind milk from either breast and on a second time point to complete expression of both breasts. Samples were collected in their homes in disposable bottles (2 × 80 mL/2 × 250 mL) using a manual breast pump  and stored at −20 °C. The samples were transported in a bag with an ice pack and stored at −80 °C.HM analysis were carried out in one mixed sample of a full expression of right and left breast. Macronutrient and energy content was analyzed by mid-infrared spectroscopy using the HM analyzer . The analysis was performed according to manufacturer’s instructions. The milk was heated to 40 °C and homogenized by ultrasonic processing before injected into the system.n = 4). The detection limit of this assay was 0.016 ng/mL. Milk lactoferrin and secretory immunoglobulin A (sIgA) were measured with a sandwich ELISA method as previously described by Czank et al. [Biochemical analyses of milk leptin, adiponectin, lysozyme, sIgA and lactoferrin were carried out at the University of Western Australia. Milk adiponectin concentration was determined by Enzyme-Linked Immunosorbent Assay (ELISA) kits . Milk leptin (m-leptin) concentration was determined by the R&D systems leptin DuoSet ELISA kit . The recovery of a known amount of leptin added to the milk samples was 97 ± 3.0%  were taken at 5 and 9 mo visits. Local anesthetic patches  and instructions were given to the parents before first visit. If possible, infants were fasting 2 h before blood samples were taken and the time, amount and composition of last meal were recorded. Methods for blood analyses are described in Information on parental characteristics and feeding practice was collected via interviews and questionnaires at 5 and 9 mo visits for both groups and at 18 mo visit for the HW-group. Appetite traits were measured at the 5 mo visit using the Baby Eating Behavior Questionnaire (BEBQ) . BEBQ inp-value < 0.05. Parametric descriptive results are presented as means and SD, or mean and range. Independent t-test was used to test differences between the characteristics of the two study groups. Differences between groups for the anthropometric measures and blood parameters adjusted for sex  and fasting time (glucose and insulin) were analyzed by general linear models. Non-normally distributed variables are presented as medians with interquartile range (IQR: 25th and 75th percentile), and Mann-Whitney U test used for comparing the study groups. Categorical variables are presented as number and percentage and comparisons were performed using Fisher’s exact test. Associations between the different macronutrients, between macronutrients/hormones and WAZ, BAZ and LAZ change, and between mothers BMI and milk composition were investigated by linear regression, for the groups separately and combined. Statistical analyses were carried out using STATATM12 . Significance was defined as In the HW-group 17 infants were referred according to the WAZ inclusion criteria. Four infants were excluded; two because they were long and had a BAZ < 2.00 and two because they had a relatively low weight gain from birth to 5 mo (both with birthweight > 4.00 kg and with a change in WAZ from birth to 5 mo of −0.16 and +0.6). Thus, the HW-group included 13 infants. Forty-two parents showed interest in participating in the NW-group and of these, 19 mother-infant dyads fulfilled the inclusion criteria. However, two infants were excluded; one had a birth weight of 2.67 kg (WAZ −1.3 SDS) followed by a catch-up with an increment in WAZ of 1.8 SDS and one had a birth weight of 4.66 kg (WAZ +2.5 SDS) followed by a catch down in WAZ of −2.9 SDS. Thus, the NW-group included 17 infants.p = 0.006) and were 1.3 cm longer (p = 0.045) at birth than the NW-group (p = 0.025) (Infants in the HW-group weighed on average 450 g more (NW-group  and BAZ = 0.025) . From birth to the 5 mo visit weight gain was excessive in the HW-group . Mean wez-scores were significantly higher in the HW-group with the exception of triceps skinfold at 9 mo  and percentage FM (%FM), as measured by BIA, were significantly higher in the HW-group at both 5 and 9 mo (both p = 0.082). Two mothers were obese (BMI ≥ 30), both in the HW-group and four fathers were obese, two in each group.Mothers in the HW-group were significantly heavier pre-pregnancy and at 5 mo . BMI wasp = 0.29). At the 9 mo visit, 83% of the HW- and 94% of the NW-group were still breastfed. At the 18 mo visit two infants in the HW group were still breastfed. The mean duration of any breastfeeding among the remaining ten mothers in the HW-group was 13.3 mo (range 8.2–18.0 mo).The mean duration of exclusive or full breastfeeding for the HW- and NW-group was 5.14 mo (range: 3.69–6.46) and 5.54 mo (range: 3.46–6.46), respectively . At the 5 mo visit, 88.5% of the infants received small amounts of complementary food and the median contribution to the total energy intake was not different in the HW-group compared to the NW-group . Milk intake data at 9 mo were incomplete and therefore not included.Although the mean 24 h milk intake was 130 g (15%) higher in the HW-group the difference was not significant (p > 0.35) or frequency of breastfeeding at 5 mo .There was no difference in milk fat, protein, lactose or energy content between the groups at 5 or 9 mo . There wp = 0.024). Twenty-four h milk intake (p = 0.01). Frequency of breastfeeding was not associated with either m-leptin intake (p = 0.23) or m-leptin concentration (p = 0.31) in the combined group.Median m-leptin content at 5 mo was significantly lower (~40%) in the HW-group . At 9 mok intake  was negap = 0.86).Milk adiponectin content was not different between the two groups at 5 and 9 mo  and therp < 0.001) and borderline higher at 9 mo (p = 0.057). For each increase in BMI unit leptin content increased 0.017 ng/mL at 5 mo. For mothers in the HW-group m-leptin at 5 mo was also positively associated with BMI , but not in the NW-group though the direction was the same (p ≥ 0.28). M-leptin at 5 mo was positively associated with maternal BMI (p < 0.001) and negatively with 24 h milk volume (p = 0.006) in a multiple regression analysis.In the combined group m-leptin content was higher in mothers with higher BMI at 5 mo (z-scores (LAZ)) or change in these z-scores from birth to the 5 mo visit .At 5 mo there were no significant associations between milk concentrations of macronutrients, energy and hormones and infant’s anthropometry  and 88% (n = 15) of the mothers in the HW-group and NW-group, respectively. During the first 14 days 15% (n = 2) infants in the HW-group and 12% (n = 2) in the NW-group received small amounts of supplemental formula, but were EBF thereafter.The initiation of breastfeeding was without any problems for 85%  (Median (IQR); 14.1 h  vs. 15.8 h  (p = 0.004). The number of night time awakenings were similar between groups (HW:median (IQR) 2  vs. NW:3 , respectively; p = 0.10).At 5 mo maternal reported infant 24 h sleep duration was 1.7 h shorter in the HW-group compared to the NW-group  though only 25% higher. The reduction in s-leptin from 5 to 9 mo in the HW-group was 62% compared to a 33% decrease in the NW-group . There was no correlation between s-leptin and m-leptin at either 5 or 9 mo (both p > 0.62).Infant serum leptin (s-leptin) was significantly higher, about double, in the HW-group compared to the NW-group at 5 mo  and FM . At 9 mo only the association between s-leptin and WAZ was still significant . There were no associations between s-leptin and LAZ at either 5 or 9 mo (p > 0.31). S-leptin in the combined group at 5 mo was negatively associated with the change in WAZ from 5 to 9 mo (p = 0.0015), and the association was also significant for the HW-group alone  and urea lower (p = 0.025) in the HW-group . Interestingly, the two groups had almost the same gain in weight and length from the 5 mo to the 9 mo visit; a gain not very different from the median in the WHO growth standard velocities [The weight gain of infants in the HW-group was excessive. The mean weight gain per week was about 60% higher than in the NW-group, which had a weight gain close to the median weight gain in the WHO growth standard . Catch-uz-scores ,27 and tlocities . From thlocities ,29,30. Hlocities , while olocities . The risz-scores from 5 to 18 mo in the HW group, %FM and fat mass index (FMI) continued to be high up to 18 mo, whereas the difference in fat-free mass index appeared to be decreasing. The medium-term effects of EWG therefore appear to manifest in both elevated length and adiposity. Links between accelerated linear growth and adiposity have been reported previously in the early post-natal period [With a body FM of 3.6 kg at 5 mo, which is 70% more than the NW-group, the rate of fat deposition from birth to 5 mo must have been much greater in the HW-group. Although there was some decrease in BMI and skinfold l period  and mighl period , resultiThe significant higher birth weight in the HW-group might also be a result of a different intra-uterine environment, partly because of the higher maternal pre-pregnancy weight in the HW-group. However, we find it unlikely that these differences between the groups could have a significant role in explaining the very large differences in post-natal growth between the groups.p = 0.19), the intake was 15% higher in the HW-group. If there has been a difference of this magnitude throughout the first 5 mo it is likely to have an effect on growth. From 5 to 9 mo weight gain in the two groups was almost identical, and therefore the energy requirements might not have been different when milk intake was measured. Ideally, measurement of milk volume should have been carried out earlier e.g., between 1 and 4 mo of age when growth velocity was considerably higher; however, recruitment precluded this. Another reason that we did not find a significant difference could be the limited power in the study. In three of the cases reported earlier, 24 h milk intake at age 4 to 5.5 mo was high; 1123, 1132 and 1421 mL [HM intake is a strong driver of infant growth ,35. Ther 1421 mL . MothersLeptin influences the regulation of appetite by inducing satiety  providinLower levels of m-leptin content have been associated with greater infant length at 1 mo postpartum, and FM at 6 mo in EBF infants , althougLow m-leptin levels may lead to higher milk intakes as a result of greater suckling pressure (frequency) in the HW-gain group. This phenomenon is generally associated with maternal malnutrition and infant catch-up growth ,40 reducMean infant s-leptin values were markedly higher in the HW-group at both 5 and 9 mo. This can be explained at least partly by the higher FM in the HW-group, as s-leptin is mainly produced by fat tissue ,37. WithA small group of infants have been shown to have very high leptin levels during the first 6 mo, with a value for the 90th percentile about four times as high as the median (11.86 vs. 2.81 ng/mL) . Our valLeptin is considered an anorexigenic hormone but it has been suggested that during early infancy some infants are likely to develop leptin resistance. It has been suggested, based on studies with mice, that high s-leptin levels, during an early postnatal leptin surge, can influence the hypothalamus and thus increase appetite and thereby growth ,51,52. TWe do not know the s-leptin levels of the HW infants when weight gain was very high during the first postnatal months. However, if leptin levels were very high and leptin resistance had developed it could have an effect on milk intake and growth. The high s-leptin levels at 5 mo could also be a result of the high FM. Our data, showing a highly significant negative association between s-leptin levels at 5 mo and weight gain from 5 to 9 mo, could be a result of decreased leptin resistance when other foods were introduced, with s-leptin then having an anorexigenic effect.Overall, it is remarkable that in this relatively small cohort there were significant differences in both m-leptin and s-leptin levels, suggesting that leptin could have a role in the excessive weight gain in the HW group. However, the potential mechanism we have discussed here are speculative and there is a need for larger studies to investigate the potential role of leptin in excessive weight gain in exclusively breastfed infants.Other potential factors that could have an effect on the high weight gain in the HW-group are the effect of milk composition on the microbiota. Both HM oligosaccharides (HMO) and leptin have been shown to be independently associated with breastfed infants gut bacteria, which could have an effect on intestinal energy harvest ,53,54. IA limitation is that it was not possible to identify, recruit, and arrange clinical visits before the age of 5 to 6 mo, by which time growth velocity was decreasing and some infants had started on complementary foods. If we had been able to measure milk intake when growth velocity was considerably higher, assessment of milk intake, milk composition and blood would most likely have given a better understanding of why these infants experience such a high weight gain. Furthermore, the power of the study was not very high due to the relatively small number of infants included. We have made multiple comparisons, but as the number of infants in the groups was small and as we have had an explorative approach, we have not controlled for this.Aside from these limitations, this study is the first to bring valuable knowledge for future studies designed to investigate the causes of EWG in exclusively breastfed infants further. A strength of the study is, that it is the first cohort of breastfed infants with EWG during the first 6 mo followed by catch-down, which has been followed longitudinally with detailed measurements of growth, body composition, HM intake and blood samples.In conclusion, our exploratory study suggests that leptin could influence the early EWG and later catch-down in the HW-group but the mechanisms are not known. There is a need to explore this in future studies, especially to understand how leptin is influencing early postnatal growth. The very high weight gain in the HW-group might increase the risk of later overweight and obesity and metabolic complications. The mean BAZ value was within the higher normal range at 18 mo, but three infants still had a BAZ above +2. Thus, it seems like at least some of the infants with a very high infant weight gain reach a weight within the normal range within the following years. In that perspective, it is interesting that in a large study, measuring BMI at 8 to 10 years, there was a highly significant inverse association between s-leptin values in infancy and childhood BMI suggesting a programming effect . Another


Background: Mucosal mast cells (MC) are key players in IgE-mediated food allergy (FA). The evidence on the interaction between gut microbiota, MC and susceptibility to FA is contradictory.Objective: We tested the hypothesis that commensal bacteria are essential for MC migration to the gut and their maturation impacting the susceptibility to FA.Methods: The development and severity of FA symptoms was studied in sensitized germ-free (GF), conventional (CV), and mice mono-colonized with L. plantarum WCFS1 or co-housed with CV mice. MC were phenotypically and functionally characterized.Results: Systemic sensitization and oral challenge of GF mice with ovalbumin led to increased levels of specific IgE in serum compared to CV mice. Remarkably, despite the high levels of sensitization, GF mice did not develop diarrhea or anaphylactic hypothermia, common symptoms of FA. In the gut, GF mice expressed low levels of the MC tissue-homing markers CXCL1 and CXCL2, and harbored fewer MC which exhibited lower levels of MC protease-1 after challenge. Additionally, MC in GF mice were less mature as confirmed by flow-cytometry and their functionality was impaired as shown by reduced edema formation after injection of degranulation-provoking compound 48/80. Co-housing of GF mice with CV mice fully restored their susceptibility to develop FA. However, this did not occur when mice were mono-colonized with L. plantarum.Conclusion: Our results demonstrate that microbiota-induced maturation and gut-homing of MC is a critical step for the development of symptoms of experimental FA. This new mechanistic insight into microbiota-MC-FA axis can be exploited in the prevention and treatment of FA in humans. Food allergy (FA) is a widespread pathological immune reaction which is initiated by generally harmless food antigens. Its global prevalence has been increasing since the 1960s, especially in industrialized countries, suggesting environmental factors play a key role in the susceptibility and etiology of this disorder . The humThe hygiene hypothesis suggests that changes in the commensal microbiota composition and/or function, due to excessive antibiotic use or increased hygiene, can increase the level of allergic sensitization , 8. By uLactobacillus plantarum is an extremely versatile lactic acid bacterium that has been isolated from a variety of habitats, such as plants, the gastro-intestinal tracts of human and animals as well as raw or fermented dairy products . Experimental animals were obtained by mating of female and male BALB/c mice and their female offspring were weaned at day 28 postnatally. Female offspring were co-housed together until day 60, when they were assigned either to the CV/OVA or CV/Ctrl group, which were housed separately thereafter. Ex-germ-free (exGF) mice were obtained by co-housing of 28-day old GF mice with age- and gender-matched CV mice. GF mice were mono-associated with Lactobacillus plantarum WCFS1 (Lp) and the level of bacterial colonization was evaluated weekly by plating serial dilution of feces on de Man, Rogosa, and Sharpe  agar plates as described previously  BALB/c mice were derived from the conventional BALB/c mice by Cesarean section and kept under axenic conditions in Trexler-type plastic isolators for at least 5 generations. The sterility was controlled as previously described . Brieflyeviously . Colonizi.p. within two-week interval with 60 μg of OVA  together with 100 μl Alu-Gel-S  adjuvant and PBS in a final volume of 200 μl on day 1 and 14. Control mice received 100 μl of PBS mixed with 100 μl of Alu-Gel-S. Two weeks after the second i.p. sensitization, mice were challenged 8 times at 2–3 day intervals (days 28–44) by i.g. gavages of 15 mg OVA in a final volume of 150 μl (OVA groups). Control mice received 150 μl of PBS by i.g. gavages (ctrl groups). Diarrhea occurrence was monitored for 30–60 min after each i.g. exposure. The diarrhea score was assessed according to the following criteria: 0–normal, well-formed stool, 1–soft, sticky well-formed stool, 2–not formed stool, 3–liquid diarrhea, 4–more than two episodes of liquid diarrhea after the antigen gavage during the treatment period. The temperature was measured by Thermocouple Thermometer with mouse rectal probe  30 min after the last i.g. exposure  and images were obtained by Fluorescent Image Analyser FLA-7000 .Total DNA from 150 mg cecal content was isolated by ZR Fecal DNA kit according to manufacturer‘s instructions . The purity, integrity and concentration of nucleic acids were confirmed by agarose gel electrophoresis and UV spectrophotometry as previously described . BacteriBlood samples were collected at sacrifice and serum was collected after centrifugation. OVA-specific serum IgE, IgG1, IgG2a, and IgA levels were determined by ELISA . Briefly5/well) were cultured in a flat-bottom 96-well plate  without any stimuli or in the presence of OVA (100 μg/well) for 72 h . Supernatants were collected and stored at −40°C until analyses. Levels of IL-4, IL-5, IL-10, IL-13, and IFN-γ were determined by the MILLIPLEX MAP Mouse Cytokine/Chemokine Panel  according to manufacturer's instructions and analyzed with the Bio-Plex System . Values are reported in pg/ml after subtraction of baseline levels of non-stimulated cell cultures.At sacrifice, spleens were aseptically removed and single-cell suspensions were prepared in RPMI-1640 containing 10% fetal bovine serum  and 1% Antibiotic-Antimycotic solution (Sigma-Aldrich). Cells  supplemented with 0.5% Triton X  was added to jejunum samples in the ratio 9:1 (w/w). After cooling on ice, the jejunum was homogenized for 1 min/40 Hz using Tissue Lyzer and stainless steel beads 7 mm , frozen in liquid nitrogen, thawed, and homogenized again. Supernatants were collected after centrifugation and stored at −80°C. Protein content of the homogenates was determined by the Pierce™ BCA Protein Assay Kit  using albumin as a standard. Levels of mouse mast cell protease-1 (MCPT-1) in serum and jejunal homogenates was determined by commercial kit Ready-SET-Go!®  according to manufacturer's instructions. Levels of IL-4, IL-13, and TNF-α in jejunal homogenates were measured by the MILLIPLEX MAP Mouse Cytokine/Chemokine Panel  according to manufacturer's instructions and analyzed with the Bio-Plex System . MCPT-1 and cytokine levels in jejunal homogenates are represented per 1 mg of total protein.Naive CV and GF BALB/c mice were euthanized by isofluran and peritoneal cavity lavage was performed twice with 5 ml of cold PBS containing 0.1% sodium azide and 0.2% gelatin from cold water fish skin (PBS-gel) . Small intestine mononuclear cells were isolated according to Scott et al.  with min6/well) were blocked for 10 min at 4°C in dark by 20% rat heat-inactivated serum in PBS containing 0.1% sodium azide. Staining was performed with fluorochrome labeled anti-mouse monoclonal Abs: CD45-FITC , FcεRIα-phycoerythrin , and CD117(c-kit)-APC-eFluor® 780  according to the manufacturer's recommendation. After 30 min cells were washed four times by cold PBS-gel and data were acquired by FACSCalibur  or FACS Aria III  flow cytometer. Analysis was performed using FlowJo software .Cells  or PBS alone (10 μl per paw) into hind footpad of naive GF and CV mice. Changes in paw width were measured with digital caliper  at time 0, 30, and 60 min. The baseline paw widths (time 0) for each mouse were measured immediately after injection and subtracted from paw widths after application to calculate tissue edema.2 in the ear tissue were determined.Intestinal tissue specimens were fixed with 4% paraformaldehyde for 24 h followed by storage in 80% ethanol. Right ears were fixed in Carnoy's fluid for 30 min and transferred to 96% ethanol. Collected and fixed tissue specimens were dehydrated by using increasing concentrations of ethanol and transferred into methyl salicylate, benzene, benzene-paraffin and paraffin. Sections (5 μm) were deparaffinized in xylene and rehydrated through an ethanol to water gradient and stained for chloracetate esterase activity which is characteristic for mast cell granula. Reagent solution was prepared by mixing of 4% pararosaniline, 2 mol/l HCl, 4% aqueous sodium nitrite, 0.07 mol/l phosphate buffer (pH 6.5), and substrate solution (Naphthol AS-D chloroacetate dissolved in N-dimethylformamide) . The sections were stained by reagent solution for 30 min in the dark and counterstained with hematoxylin for 2 min. The numbers of mast cells per randomly selected villi in jejunum and per 0.1 mmJejunal tissues were stored in RNA-later reagent  overnight at 4°C and kept at −80°C until processed. Tissue samples were homogenized by Precellys 24 tissue homogenizer  at 5,000 rpm for 20 s using tubes with zirconium oxide beads. RNA was isolated via RNeasy Mini kit . An iScript cDNA Synthesis Kit  was used to generate cDNA. Real-Time (RT) PCR was performed on the LightCycler® 480 instrument  using LightCycler® 480 SYBR Green I Master according to the manufacturer's instructions . β-actin was used as an internal control to normalize gene expression using the 2–ΔCt method . RT PCR t-test. GraphPad Software was used to evaluate the data ; P < 0.05 were considered significant. Data are expressed as means ±SEM.Statistical analysis between multiple groups was performed by ANOVA with Tukey's multiple comparison test. Differences between two groups were evaluated using After intraperitoneal injection of OVA adsorbed to alum, CV and GF mice were challenged by oral gavage of OVA eight times over the period of 2 weeks . Sensitiex vivo  is a key factor in MC biology . Besides by FACS . To func. Here we show that sensitization and challenge lead to increased OVA-specific IgE levels in sera and increased production of OVA-specific IL-5 and IL-13 in re-stimulated splenocytes from GF mice compared to CV animals. The concept that signals derived from commensal bacteria are crucial to normalize elevated Th2 responses have been tested previously by us and others. For example, colonization of GF mice with conventional or SPF microbiota, with a well-defined mixture of different strains, or with only a single bacterial strain led to reduced levels of serum IgE and Th2 cytokine production in comparison to GF animals . Here we, 41. RecSurprisingly, despite the elevated systemic OVA-specific humoral and cellular Th2 responses, GF mice were protected from FA symptoms, i.e., they failed to develop allergic diarrhea and hypothermia. This observation is in disagreement with studies by Cahenzli et al., Rodriguez et al. and Stefka et al. who showed that GF mice are more susceptible to FA , 31, 42.Cxcr2 and its ligands in intestinal tissue. Concomitantly, challenged GF mice had significantly lower production of MCPT-1 both locally in the intestinal tissue and systemically in sera. Further experiments are required to dissect whether the microbiota impacts MC recruitment and/or function directly or indicrectly via the action on intestinal epithelial or innate lymphoid cells.To determine the discrepancy between high levels of sensitization and the absence of FA symptoms, we investigated the presence of intestinal MC, which are widely recognized key effectors of allergy in the periphery , 28, 46.Together with a previous report about the crucial role of MC for the development of FA symptoms , our datIn our model, intestinal MC numbers in sensitized and challenged GF animals reached 60% of those observed in CV animals. Yet the GF mice were fully protected from the development of allergic diarrhea and hypothermia. This indicates that not only the numbers of local MC in GF mice, but also the functionality and maturation status of MC are impaired in these mice. In a recent paper, Wang et al. clearly demonstrated that the microbiota drives the recruitment and functionality of skin MC through the LTA-TLR2-dependent production of SCF by keratinocytes . In agreIn order to test the functionality of MC we challenged the GF and CV mice by injection of degranulation compound 48/80. GF mice exhibited significantly lower swelling compared to CV animals confirming the impaired functionality and lower maturation of MC in the absence of microbiota.L. plantarum remained unresponsive to OVA challenge in contrary to CV mice. This observation is surprising, as L. plantarum WCFS1 is a bacterial strain with strong immunomodulatory properties (Finally, we could show that conventionalization rendered exGF mice sensitive to FA, as demonstrated by hypothermia, diarrhea, and elevated levels of MCPT-1 in the gut and serum. Interestingly, mice mono-colonized with Gram-positive strain operties , 17, 51 operties . Neverthoperties , 53. WheTaken together, we report here that commensal bacteria impact MC migration and maturation in the intestine, thus playing a key role in the susceptibility to food-induced allergy. Our model based on CT-free systemic sensitization and oral challenge leads to full development of hypothermia and diarrhea in CV settings but not GF mice, represents an important tool to investigate the role of microbiota in the development or prevention of infectious or immune-mediated inflammatory diseases. The mechanistic insight into the role of the commensals-MC-FA axis, with a focus on the microbiota-induced recruitment and maturation of MC in the intestinal mucosa, can pave the way to the design of novel strategies for the prevention and treatment of food allergy in humans.MSch, LT, HK, and IS conceived and designed the experiments. MSch, PH, JG, CZ, TH, DS, and JA performed the experiments. MSch, PH, JG, CZ, TH, MS and DS analyzed the data. MSch, PH, UW, LT, HK, and IS wrote the paper. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.